Method for modulating responsiveness to steroids

ABSTRACT

A method for enhancing steroid efficacy in a steroid refractory patient afflicted with an inflammatory condition not responding or responding poorly or inadequately to anti-inflammatory treatment comprises administering an oligonucleotide having the sequence formula 
                         (SEQ. ID. No. 18)         5′-X m -TTCGT-Y n -3′                
in an effective amount to said patient and wherein X is A, T, C or G, Y is A, T, C or G, m=0-7, n=0-7 and wherein at least one CG dinucleotide is unmethylated.

RELATED APPLICATIONS

The present application is a continuation of and claims priority under35 U.S.C. 120 of U.S. application Ser. No. 11/917,748 filed Jun. 9,2008, now U.S. Pat. No. 8,148,341 which is a 371 of PCT/SE2006/050232filed Jun. 30, 2006 and claims priority under 35 U.S.C. 119 of U.S.application Ser. No. 60/696,050 filed Jul. 1, 2005.

FIELD OF THE INVENTION

The present invention relates to a method for modulating responsivenessto steroids in a patient. In particular, the invention relates a methodto reverse steroid resistance or improve steroid responsiveness in apatient thereby allowing the subject to be treated with steroids suchthat steroids reach the desired anti-inflammatory effect. The presentinvention also relates to the use of the oligonucleotides used in theinventive method for the manufacture of a medicament.

BACKGROUND

Inflammation is a complex disease involving many factors and cell types.From a disease perspective, many years of research have taught us thatinflammatory disorder such as asthma, rheumatoid arthritis, ulcerativecolitis, and Cohn's disease and others have a distinct inflammatorycytokine profile. These profiles are the result of the nature of theresponding lymphocytes. In other words, inflammation cannot beconsidered as just “inflammation” but rather that different inflammatorydiseases are associated with different secreted cytokines that enhancethe proliferation and differentiation of certain sub-populations of Thelper cells.

The nature and magnitude of an immune response is largely dictated bythe profile of the foreign antigen to which the immune system has beenexposed. This event sets into motion a series of events that ultimatelyleads to the generation of humoral and cell-mediated immunity. These twodifferent effector functions are brought about by the presence of twosubpopulations of helper T cells. As also indicated, differentinflammatory diseases can be segregated as being either Th1 or Th2,depending on the cytokine profile seen.

Under “normal” healthy conditions there is a delicate balance betweenpro-inflammatory cytokines typical of Th1 and anti-inflammatorycytokines typical of Th2. If this balance is lost, there will be apolarization resulting in predominantly Th1 or Th2 type inflammation andclinical manifestation of the disease will occur.

Some newer forms of therapeutics now attempt to restore the “in-balance”in for example Th1 type diseases by reducing the cytokine profile of Th1and thereby allow more of a Th2 profile to occur (Neurath et al, 1995;Mannon et al, 2004). Over the last 5 years or so, many researchers havedemonstrated both in vitro and in vivo the validity of the use ofoligonucleotides as immunostimulatory agents in immunotherapyapplications. The observation that phosphodiester and even modifiedphosphorothioate oligonucleotides can induce immune stimulation hascreated a growing interest in developing this effect as a therapeutictool.

Bacterial DNA has immune stimulatory effects capable of activating Bcells and natural killer cells, but vertebrate DNA does not (reviewed inKrieg, 1998, Applied Oligonucleotide Technology, C. A. Stein and A. M.Krieg, (Eds.), John Wiley and Sons, Inc., New York, N.Y., pp. 431-448).It is now understood that these immune stimulatory effects of bacterialDNA are a result of the presence of unmethylated CpG dinucleotides, inparticular base contexts (CpG motifs), which are common in bacterialDNA, but methylated and underrepresented in vertebrate DNA (Krieg et al,1995). The immune stimulatory effects of bacterial DNA can be mimickedwith synthetic oligodeoxynucleotides (ODN) containing these CpG motifs.Such CpG ODN have highly stimulatory effects on human and murineleukocytes, inducing B cell proliferation; cytokine and immunoglobulinsecretion; natural killer (NK) cell lytic activity and IFN-gammasecretion; and activation of dendritic cells (DCs) and other antigenpresenting cells to express costimulatory molecules and secretecytokines, especially the Th1-like cytokines that are important inpromoting the development of Th1-like T cell responses. These immunestimulatory effects of native phosphodiester backbone CpG ODN are highlyCpG specific in that the effects are dramatically reduced if the CpGmotif is methylated, changed to a GpC, or otherwise eliminated oraltered (Krieg et al, 1995 and Hartmann et al, 1999).

In early studies, it was thought that the immune stimulatory CpG motiffollowed the formula purine-purine-CpG-pyrimidine-pyrimidine (Krieg etal, 1995; Pisetsky, 1996 and Hacker et al., 1998).

Currently there is a significant amount of published data indicatingthat oligonucleotides containing CpG motifs induce certain cytokines,for example, human and mouse cells respond to CpG motif oligonucleotidesby enhanced secretion of interferon-gamma (IFN-gamma) (Iho et al., 1999:Cowdery et al., 1996) IL-1, IL-6, TNF-alpha and IL-12 (Stacey et al.,1996; Jakob et al., 1998 and Sparwasser et al., 1998).

Due to the nature of cytokines induced, CpG containing oligonucleotidesare largely considered to induce a Th1 profile both in vitro and in vivo(Zimmermann et al., 1998; Kline, 2000).

In addition to the presence of CpG motifs, researchers have also notedthat synthesizing oligonucleotides with a full nuclease-resistantphosphorothioate (PS) backbone can potentate the stimulatory effects ofthe oligonucleotides, in that these oligonucleotides were much morepotent at stimulating B cells, whereas the same sequence with nativephosphodiester backbone had no effect (Zhao et al., 1996).

While the presence of a CpG motif within the sequence of anoligonucleotide can induce a strong Th1 cytokine response, this responseshould be considered in the overall context of the compounds state ofchemical modification as well as the general sequence structure.

As already indicated in the background introduction to inflammation,there is a specific cytokine profile that becomes prominent in varioustypes of inflammatory diseases. For example in asthmatic patients thereare high levels of IL-4 and low levels of IFN-gamma. This cytokinepicture would indicate that asthma is a Th2 type of disease. Rheumatoidarthritis by contrast is better associated with a Th1 type ofinflammation characterized in that high levels of IFN-gamma and lowerlevels of IL-4 are seen.

The phenomenon of corticosteroid resistance has been most extensivelystudied in asthmatic patients and to a lesser degree in ulcerativecolitis where evidence over the years has accumulated, pointing to anumber of cytokine abnormalities. Both diseases are classified as Th2type and interferons as well IL-10 have been implicated as beingimportant factors in the pathogenesis of corticosteroid resistance.

It may be possible that immunostimulatory oligonucleotides that are ableto induce endogenous production of such cytokines, such as interferonsand IL-10, are able to influence the inflammatory status of setroidresistance or steroid dependent patients in a beneficial manner.

The evidence that certain cytokines can influence the steroidresponsiveness is gathered from clinical studies conducted incorticosteroid resistant asthmatic and ulcerative colitis patients whowere also all on corticosteroid therapies. In fact, this type of patientsubgroup characteristic was the only common denominator between theclinical studies described below.

Interferons (IFNs) play crucial roles in the regulation of a widevariety of innate and adaptive immune responses. Type I interferons(IFN-alpha/beta) are central to the host defense against pathogens suchas viruses, whereas type II interferon (IFN-gamma) mainly contributes tothe T-cell-mediated regulation of the immune responses (Taniguchi andTakaoka, 2001). Interferons have also found their place in thesuccessful treatment of various human diseases such as benign neoplastic(Gill et al, 1995) and viral diseases (Niederau et al, 1996; Zeuzem etal, 2000).

In a study (Simon et al, 2003), 10 patients with corticosteroidresistant asthma where administered IFN-alpha (3×10⁶ IU/day) (Roferon A®Roche) in addition to the prednisone dose they were all receiving. Thetrial demonstrated high efficacy in these patients and clinical signs ofimprovement occurring 1-2 weeks after cytokine therapy, allowing thedose of corticosteroids to be reduced. The authors further noted thatthe IFN-alpha treatment increased the capacity of peripheral blood Tcells to produce IFN-gamma, suggesting there had been a shift from a Th2type response (typical of asthma and allergic diseases) to a Th1response.

Moreover, the authors showed that there was also an increase in blood Tcells secreting IL-10 in those patients that had received cytokinetherapy. As corticosteroids mediate their anti-inflammatory effects, inpart, by increasing levels of IL-10, the authors conclude thatadministration of exogenous IFN-alpha broke the corticosteroidresistance in these patients.

Musch et al (2002) demonstrated a high response rate in corticosteroidrefractory ulcerative colitis patients when given IFN-beta i.v. Thepilot study enrolled 25 severely ill ulcerative colitis patients provingrefractory to basic medication. All patients where on corticosteroids atthe time of cytokine treatment. Following treatment, 22 of the 25 (88%)went into remission within 3 weeks with a strong decrease in clinicalactivity index (CAI) noted 1 week after initiating treatment. The meanlength of response was 13 months.

In another study, Sumer et al, (1995), reported an 82% improvement rateto s.c. IFN-alpha cytokine treatment in corticosteroid resistantulcerative colitis patients. They further noted that the 23 patientsresponded to the cytokine therapy with a fast improvement (within 15days) and were in complete clinical and endoscopic remission after 6months of therapy. Three patients entered remission after longertherapy; however, all 26 patients were observed for more than 2 yearswithout receiving additional therapy and remained in full clinical andendoscopic remission during this period.

Another cytokine that has received interest in the pathogenesis ofcorticosteroid resistance is IL-10. This cytokine is believed to havepotent anti-inflammatory effects in that it can suppress the productionof pro-inflammatory cytokines. It also has broad implications in thedevelopment of certain inflammatory diseases, most noticeably allergyand asthma (Hawrylowicz et al, 2005), as well as playing a central rolein the regulation of immune responses. It is believed thatcorticosteroids exert their anti-inflammatory effects in part byenhancing IL-10 production (Richards et al, 2005).

Numerous clinical studies have indicated that there is a general lack ofsufficient levels of IL-10 in asthmatics which may potentiallycontribute to a more intensive inflammation. In a randomizeddouble-blind clinical study conducted in children with moderate atopicasthma, Stelmach et al (2002) demonstrated that treatment withTriamcinolone, a corticosteroid, and montelukast, an anti-leukotriene,significantly increased levels of IL-10 in blood serum and in additionsignificantly improved clinical symptoms.

In another clinical study, levels of IL-10 and IL-10 producing cellswere shown to be significantly reduced in patients with severepersistent asthma when compared to mild asthma (Tomitai et al, 2002).These observations were in agreement with previous findings that thereis a defect in the production of cells that are able to produce IL-10 inasthmatic subjects (Tormey et al, 1998).

This defect was also shown to exist in corticosteroid resistantasthmatic patients. Under normal conditions, corticosteroids will causean increased production of IL-10 in corticosteroid sensitive patients.However, Hawrylowicz et al (2002) could confirm that in corticosteroidresistant asthmatic patients, corticosteroids failed to induce IL-10synthesis. These observations suggest a strong link between induction ofIL-10 synthesis and efficacy of corticosteroids.

In a recently published study (Xystrakis et al, 2006), the authorsisolated PBMC from corticosteroid resistant asthmatic patients and coulddemonstrate that addition of vitamin D3 with dexamethasone to thesecultures enhanced IL-10 synthesis to levels observed in cells fromcorticosteroid sensitive patients cultured with dexamethasone alone.Furthermore, and perhaps most significantly, pre-treatment with IL-10fully restored IL-10 synthesis in these cells in response todexamethasone.

The use of human bacterial flora to treat gastrointestinal (GI)disorders is not a novel concept, having been practiced periodically formore than 40 years (Eiseman et al, 1958). Significant clinicalimprovements have been observed in numerous GI disorders includinginflammatory bowel disease (IBD) (Bennet and Brinkman 1989). Borody etal, reported in 2003 that human bacteriotherapy could be used to treatsevere corticosteroid resistant ulcerative colitis (UC).

In a small study, 6 chronic UC patients who had all previously failedmaximum tolerated standard corticosteroid therapies were all given asingle fecal enema concomitant to corticosteroid therapies they werecurrently on. Complete reversal of UC was achieved in all 6 patientsfollowing the rectal infusion. The authors also state that all patientsceased anti-inflammatory therapy within 6 weeks and remained inremission in one case for up to 13 years. The apparent success ofbacteriotherapy in resistant ulcerative colitis patients may be due tothe repopulation of the colon with a “healthy” bacterial flora, butequally as the authors suggest, may also be due to the instillation of alarge amount of bacterial DNA, containing abundant CpG motifs, whichinduced a beneficial immunomodulating effect resulting in completereversal of the disease.

A study in asthmatics compared the response to a steroid (prednisone) inboth steroid resistant and steroid sensitive patients. The patients werefirst given a “wash-out” period of one week before administration of thesteroid. Cytokine profiles before administration and 1 week afterindicated that those patients that responded to the steroid moved from aTh2 type to a more Th1 like status. By contrast, those patients thatfailed to respond to the administered steroid remained Th2 type (Naseeret al., 1997).

While the reason for steroid resistance in asthmatic patients is notentirely clear, numerous studies in humans have indicated that thosepatients that are resistant to steroids have high persistent levels ofIL-2/4 that are not suppressed by the action of steroids. Furthermore,in vitro studies indicate that when IL-2/4 is placed in the culturemedium, the cells become resistant to the action of steroids (Sousa A Ret al., 2000; Hamid Q A et al., 1999).

In rheumatoid arthritis a similar scenario has been suggested in thatsteroid resistant patients demonstrate high levels of IL-4, which cannotbe reduced when challenged with steroids (Chikanza et al., 2004). Ofinterest are the findings that IFN-gamma is able to down regulate IL-4responses (Eui-Young et al., 2000; Smeltz et al., 2002) at the level oftranscription.

Steroid resistance or dependence is still a major clinical concern for alarge number of patients afflicted with inflammatory diseases as currenttherapies rely on the use of potent immunomodulators that can induceserious side-effects. A simple straightforward method to enhance steroidefficacy in a steroid unresponsive individual with little risk ofunwanted side-effects would essentially improve anti-inflammatorytreatment, thus ameliorating the disease in question, and increasing thequality and length of life for a large number of patients.

SUMMARY OF THE INVENTION

The present invention relates to the surprising discovery of a methodfor enhancing steroid efficacy in a steroid refractory or steroiddependent patient afflicted with an inflammatory condition notresponding or responding poorly or inadequately to anti-inflammatorytreatment or there is an inability to wean the anti-inflammatorytreatment dosing level down. An oligonucleotide having the sequence5′-X_(m)-TTCGT-Y_(n)-3′ (SEQ. ID. No. 18) is administered in aneffective amount to said patient and wherein X is A, T, C or G, Y is A,T, C or G, m=0-7, n=0-7 and wherein at least one CG dinucleotide isunmethylated.

The present invention also relates to the use of the above mentionedoligonucleotides for the manufacture of a medicament for enhancingsteroid efficacy in a steroid refractory patient afflicted with aninflammatory condition not responding or responding poorly orinadequately to anti-inflammatory treatment.

The attached set of claims is hereby incorporated in its entirety.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the number of IL-10 producing cells inresponse to 48 hrs of DIMS0150 stimulation in PBMC from five (n=5)different healthy donors analysed by ELISpot. PBMC were incubated inmedium (basal) or with increasing concentrations (0.1, 1, 5, 10, 25,100, 150 or 200 μM) of the CpG containing DIMS0150 or, or its GpCcontrol IDX0526, or the CpG ODNs, IDX0910 (0.1 or 10 μM) and IDX0900 (3μM) for 48 hours before detection of IL-10 positive spots. Each bar ofthe histogram represents the average results from five different blooddonors. Samples were performed and analysed in triplicate for eachexperiment/blood donor. Note that IDX0900 was tested on threeindividuals (n=3).

FIG. 2 is a graph showing the number of IFN-gamm

producing cells in response to 72 hrs of DIMS0150 stimulation of PBMCfrom five (n=5) different donors as analysed by ELISpot. PBMC wereincubated in medium (basal) or with increasing concentrations (0.1, 1,5, 10, 25, 50 100, 150 or 200 μM) of the CpG containing DIMS0150, or itsGpC control IDX0526, or the CpG ODNs IDX0910 (at 0.1 μM) and IDX0900 (at3 μM) for 72 hours before detection of IFN-gamma positive spots. Eachbar of the histogram represents the average results from five differentblood donors. Samples were performed and analysed in triplicate for eachexperiment/blood donor. Note that IDX0900 was tested on threeindividuals (n=3).

FIG. 3 is a graph showing the number of IFN-alpha producing cells inresponse to 48 hrs DIMS0150 in PBMC from ten (n=10) different healthydonors as assayed by ELISpot. PBMC were incubated in medium (basal) orwith increasing concentrations (0.1, 1, 5, 10, 25, 50, 100, 150 or 200μM) of the CpG containing DIMS0150, or its GpC control IDX0526 (n=9) orthe CpG ODN IDX0910 (0.1 μM or 10 μM) for 48 hours before detection ofIFN-alpha positive spots. Each bar of the histogram represents theaverage results from ten different blood donors. Samples were performedand analysed in triplicate for each experiment/blood donor. Note thatIDX0910 at 0.1 μM was tested on eight donors and 10 μM was tested onfour individuals.

FIG. 4A is a graph showing the IL-10 production in response to 48 hrsstimulation with DIMS0150 as quantified by ELISA. PBMC were incubatedwith increasing concentrations (0.1, 1, 5, 10, 25, 50, 100, 150 or 200μM) of DIMS0150 or its GpC control IDX0526. As controls, cells were leftin medium (basal) or treated with CpG ODNs IDX0910 (0.1 μM) and IDX0900(3 μM). This graph represents results from an experiment in PBMC fromone of two donors performed and analysed in duplicate.

FIG. 4B is a graph showing the IFN-gamma production in response to 48hrs stimulation with DIMS0150 as quantified by ELISA. PBMC wereincubated with increasing concentrations (0.1, 1, 5, 10, 25, 50, 100,150, 200 or 300 μM) of DIMS0150 or its GpC control IDX0526. As controls,cells were left in medium (basal) or treated with CpG ODNs, IDX0910 (0.1μM and 1 μM) or IDX0900 (3 μM). This experiment was performed in cellsfrom one blood donor and each sample was performed and analysed induplicate.

FIG. 4C is a graph showing the IFN-alpha production in response to 48hrs stimulation with DIMS0150 as quantified by ELISA. PBMC wereincubated with different concentrations (0.1, 1, 5, 10, 25, 50, 100,150, 200 or 300 μM) of DIMS0150 or its GpC control IDX0526. As controls,cells were left in medium (basal) or treated with CpG ODNs, IDX0910 (0.1μM and 1 μM) and IDX0900 (3 μM). This graph represents results from anexperiment in PBMC from one of two donors performed and analyzed induplicate.

FIG. 5 is a graph showing the comparison of IL-10 production in humanPBMC upon stimulation with a variety of CpG ODNs and their reversedcontrols as quantified by ELISA. PBMC were treated with increasingconcentrations (from left to right, as indicated by the triangle: 0.1,1, 10 or 100 μM) of DIMS0150, IDX0250, IDX0920 and IDX 0910 ODNs andtheir respective negative control GpC ODNs together with the non-CpGcontaining ODN IDX0304 for 48 hours before collection of supernatantsand subsequent analysis. Cells left untreated in medium exhibited thebasal level of IL-10 in PBMC. Supernatants were collected after 48 hoursfollowed by subsequent analysis. This experiment was performed on cellsfrom one blood donor and all samples were performed and analysed induplicate.

FIG. 6 is a graph showing the comparison of IFN-gamma production inhuman PBMC upon stimulation with a variety of CpG ODNs as quantified byELISA. PBMC were treated with increasing concentrations (from left toright, as indicated by the triangle: 0.1, 1, 10 or 100 μM) of DIMS0150,IDX0250, IDX0920 and IDX 0910 ODNs and their respective negative controlGpC ODNs together with the non-CpG containing ODN IDX0304 for 48 hoursbefore collection of supernatants and subsequent analysis. Cells leftuntreated in medium exhibited the basal level of IFN-gamma in PBMC.Supernatants were collected after 48 hours followed by subsequentanalysis. This experiment was performed on cells from one blood donorand all samples were performed and analysed in duplicate.

FIG. 7 is a graph showing the IFN-gamma production from mousesplenocytes in response to 48 hs of CpG-stimulation as quantified byELISA. Mouse splenocytes were treated with increasing concentrations(from left to right, as indicated by the triangles: 0.1, 1, 10 or 100μM) of DIMS0150, IDX0250, IDX0920, IDX0910 ODNs and their respectivenegative control GpC ODNs compared to the non CpG-containing ODN controlIDX0304 for 48 hours before collection of supernatants and subsequentanalysis. Cells left untreated in medium exhibit the basal level ofIFN-gamma in splenocytes. Supernatants were collected after 48 hours ofstimulation followed by subsequent analysis. Note that this experimentwas performed in cells from one mouse spleen and all samples wereperformed and analyzed in duplicate.

FIG. 8 is a graph showing the IL-10 production from mouse splenocytes inresponse to 48 hs of CpG-stimulation as quantified by ELISA. Mousesplenocytes were treated with increasing concentrations (from left toright, as indicated by the triangle: 0.1, 1, 10 or 100 μM) of DIMS0150,IDX0250, IDX0920 and IDX 0910 ODNs and their respective negative controlGpC ODNs together with the non-CpG containing ODN IDX0304 for 48 hoursbefore collection of supernatants and subsequent analysis. Cells leftuntreated in medium exhibited the basal level of IL-10 in splenocytes.Supernatants were collected after 48 hours followed by subsequentanalysis. This experiment was performed on cells from one mouse spleenand all samples were performed and analysed in duplicate.

FIGS. 9A and B shows the IL-10 release from human PBMC in response toDIMS0150 and truncated versions of SEQ.ID.NO.1 as depicted in table 1.

FIGS. 10A and B shows the IL-10 and IL-6 release from human PBMCpre-incubated with varying concentrations of chloroquine in response tostimulation with CpG compounds and controls, respectively.

DETAILED DESCRIPTION

As used herein, the terms “steroid resistant” and “steroid refractory”refers to patients having inflammatory diseases in which administrationof steroid treatment, typically effective in patients having suchdiseases, is ineffective. In this context “steroid resistant” and“steroid refractory” patients include, but are not limited to, patientswho do not respond or respond poorly or inadequately as judged by commonappropriate physiological parameters to systemic or topical administeredsteroids. Two types of steroid resistant patients have been describedi.e. acquired steroid resistance (Type I) and primary steroid resistance(Type II), both of which are comprised in the present invention.

As used herein, the term “steroid dependence”, refers to patients withthe inability to be weaned off systemic or topical administered steroidtreatment.

References describing immunostimulatory activity of polynucleotidesinclude, but are not limited to, Krug et al. (2001); Bauer et al.(2001); Klinman et al. (1999); Jahn-Schmid et al. (1999) and Tighe etal. (2000).

Further references describing immunostimulatory sequences include:Tokunaga et al. (1992); Yamamoto et al. (1992) and EP 468,520; WO96/02555; WO 97/28259; WO 98/16247; U.S. Pat. Nos. 6,339,068, 6,406,705,6,426,334 and 6,426,336.

All patents, patent applications, and publications cited herein arehereby incorporated by reference in their entirety.

For purposes of the invention, the term “oligonucleotide” refers to apolynucleoside formed from a plurality of linked individual nucleosideunits. Such oligonucleotides can be obtained from existing nucleic acidsources, including genomic or cDNA, but are preferably produced bysynthetic methods. The nucleoside residues can be coupled to each otherby any of the numerous known internucleoside linkages. Suchinternucleoside linkages include, without limitation, the naturalinternucleoside phosphodiester bond or indeed modified internucleosidessuch as, but not limited to, phosphorothioate, phosphorodithioate,alkylphosphonate, alkylphosphonothioate, phosphotriester,phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate,carbamate, morpholino, borano, thioether, bridged phosphoramidate,bridged methylene phosphonate, bridged phosphorothioate, and sulfoneinternucleoside linkages. The term “oligonucleotide” also encompassespolynucleosides having one or more stereospecific internucleosidelinkage (e.g., (Rp)- or (Sp)-phosphorothioate, alkylphosphonate, orphosphotriester linkages). As used herein, the terms “oligonucleotide”and “dinucleotide” are expressly intended to include polynucleosides anddinucleosides having any such internucleoside linkage, whether or notthe linkage comprises a phosphate group. In certain preferredembodiments, these internucleoside linkages may be phosphodiester,phosphorothioate, or phosphorodithioate linkages, or combinationsthereof.

The term “oligonucleotide” also encompasses polynucleosides havingadditional substituents including, without limitation, protein groups,lipophilic groups, intercalating agents, diamines, folic acid,cholesterol and adamantane. The term “oligonucleotide” also encompassesany other nucleobase containing polymer, including, without limitation,peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups(PHONA), locked nucleic acids (LNA), morpholino-backboneoligonucleotides, and oligonucleotides having backbone sections withalkyl linkers or amino linkers.

The oligonucleotides of the invention can include naturally occurringnucleosides, modified nucleosides, or mixtures thereof. As used herein,the term “modified nucleoside” is a nucleoside that includes a modifiedheterocyclic base, a modified sugar moiety, or a combination thereof. Insome embodiments, the modified nucleoside is a non-natural pyrimidine orpurine nucleoside, as herein described. In some embodiments, themodified nucleoside is a 2′-substituted ribonucleoside anarabinonucleoside or a 2′-deoxy-2′-substituted-arabinoside.

The term “oligonucleotide” includes hybrid and chimericoligonucleotides. A “chimeric oligonucleotide” is an oligonucleotidehaving more than one type of internucleoside linkage within its sequencestructure. One preferred example of such a chimeric oligonucleotide is achimeric oligonucleotide comprising a phosphorothioate, phosphodiesteror phosphorodithioate region and non-ionic linkages such asalkylphosphonate or alkylphosphonothioate linkages (Pederson et al. U.S.Pat. Nos. 5,635,377 and 5,366,878).

A “hybrid oligonucleotide” is an oligonucleotide having more than onetype of nucleoside. One preferred example of such a hybridoligonucleotide comprises a ribonucleotide or 2′-substitutedribonucleotide region, and a deoxyribonucleotide region (Metelev andAgrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and 6,143,881).

For purposes of the invention, the term “immunomodulatoryoligonucleotide” refers to an oligonucleotide as described above thatinduces an immune response either stimulating the immune system orrepressing the immune system or both in an organism when administered toa vertebrate, such as a mammal. As used herein, the term “mammal”includes, without limitation rats, mice, cats, dogs, horses, cattle,cows, pigs, rabbits, non-human primates, and humans.

Preferably, the immunomodulatory oligonucleotide comprises at least onenaturally occurring phosphodiester, or one modified phosphorothioate, orphosphorodithioate internucleoside linkage, however preferred linkagesor indeed backbone modifications including, without limitation,methylphosphonates, methylphosphonothioates, phosphotriesters,phosphothiotriesters, phosphorothioates, phosphorodithioates, triesterprodrugs, sulfones, sulfonamides, sulfamates, formacetal,N-methylhydroxylamine, carbonate, carbamate, morpholino,boranophosphonate, phosphoramidates, especially primaryamino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, andstereospecific linkages (e.g., (Rp)- or (Sp)-phosphorothioate,alkylphosphonate, or phosphotriester linkages).

The term “immunomodulatory response” describes the change of an immuneresponse when challenged with an immunomodulatory oligonucleotide. Thischange is measurable often through the release of certain cytokines suchas interferons as well as other physiological parameters such asproliferation. The response can equally be one that serves to stimulatethe immune system as well as to repress the immune system depending onthe cytokines induced by the immunomodulatory oligonucleotide inquestion.

In some embodiments, the immunomodulatory oligonucleotide comprises animmunostimulatory dinucleotide of formula 5′-Pyr-Pur-3′, wherein Pyr isa natural or synthetic pyrimidine nucleoside and Pur is a natural orsynthetic purine nucleoside. In some preferred embodiments, theimmunomodulatory oligonucleotide comprises an immunostimulatorydinucleotide of formula 5′-Pur*-Pur-3′, wherein Pur* is a syntheticpurine nucleoside and Pur is a natural or synthetic purine nucleoside.In various places the dinucleotide is expressed as RpG, C*pG or YZ, inwhich case respectively, R, C*, or Y represents a synthetic purine. Aparticularly preferred synthetic purine is2-oxo-7-deaza-8-methyl-purine. When this synthetic purine is in the Pur*position of the dinucleotide, species-specificity (sequence dependence)of the immunostimulatory effect is overcome and cytokine profile isimproved. As used herein, the term “pyrimidine nucleoside” refers to anucleoside wherein the base component of the nucleoside is a monocyclicnucleobase. Similarly, the term “purine nucleoside” refers to anucleoside wherein the base component of the nucleoside is a bicyclicnucleobase. For purposes of the invention, a “synthetic” pyrimidine orpurine nucleoside includes a non-naturally occurring pyrimidine orpurine base, a non-naturally occurring sugar moiety, or a combinationthereof.

In some embodiments, the sugar moiety of the nucleoside can be anon-naturally occurring sugar moiety. For purposes of the presentinvention, a “naturally occurring sugar moiety” is a sugar moiety thatoccurs naturally as part of a nucleic acid, e.g., ribose and2′-deoxyribose, and a “non-naturally occurring sugar moiety” is anysugar that does not occur naturally as part of a nucleic acid, but whichcan be used in the backbone for an oligonucleotide, for example but motlimited to hexose. Arabinose and arabinose derivatives are examples ofpreferred sugar moieties.

Preferred immunostimulatory moieties according to the invention furtherinclude nucleosides having sugar modifications, including, withoutlimitation, 2′-substituted pentose sugars including, without limitation,2′-O-methylribose, 2′-O-methoxyethyl-ribose, 2′-O-propargylribose, and2′-deoxy-2′-fluororibose; 3′-substituted pentose sugars, including,without limitation, 3′-O-methylribose; 1′,2′-dideoxyribose; arabinose;substituted arabinose sugars, including, without limitation,1′-methylarabinose, 3′-hydroxymethylarabinose,4′-hydroxymethylarabinose, 3′-hydroxyarabinose and 2′-substitutedarabinose sugars; hexose sugars, including, without limitation,1,5-anhydrohexitol; and alpha-anomers.

In another embodiment, preferred immunostimulatory moieties according tothe invention further include oligonucleotides having other carbohydratebackbone modifications and replacements, including peptide nucleic acids(PNA), peptide nucleic acids with phosphate groups (PHONA), lockednucleic acids (LNA), morpholino backbone oligonucleotides, andoligonucleotides having backbone linker sections having a length of fromabout 2 angstroms to about 200 angstroms, including without limitation,alkyl linkers or amino linkers. The alkyl linker may be branched orunbranched, substituted or unsubstituted, and chirally pure or a racemicmixture. Most preferably, such alkyl linkers have from about 2 to about18 carbon atoms. In some preferred embodiments such alkyl linkers havefrom about 3 to about 9 carbon atoms. Some alkyl linkers include one ormore functional groups selected from the group consisting of hydroxy,amino, thiol, thioether, ether, amide, thioamide, ester, urea, andthioether. Some functionalized alkyl linkers are poly (ethylene glycol)linkers of formula-0-(CH2-CH2-O—), (n=1-9). Some other functionalizedalkyl linkers are peptides or amino acids.

In a further embodiment preferred immunostimulatory moieties accordingto the invention further include DNA isoforms, including, withoutlimitation, -L-deoxyribonucleosides and a-deoxyribonucleosides.Preferred immunostimulatory moieties according to the inventionincorporate 3′ modifications, and further include nucleosides havingunnatural internucleoside linkage positions, including, withoutlimitation, 2′-5′, 2′-2′,3′-3′ and 5′-5′ linkages.

The immunomodulatory oligonucleotide according to the invention compriseat least five nucleosides linked via internucleoside linkage or afunctionalized nucleobase or sugar via a non-nucleotidic linker. Forpurposes of the invention, a “non-nucleotidic linker” is any moiety thatcan be linked to the oligonucleotides by way of covalent or non-covalentlinkages.

Non-covalent linkages include, but are not limited to, electrostaticinteraction, hydrophobic interactions, -stacking interactions, andhydrogen bonding. The term “non-nucleotidic linker” is not meant torefer to an internucleoside linkage, as described above, e.g. aphosphodiester, phosphorothioate, or phosphorodithioate functionalgroup, that directly connects the 3′-hydroxyl groups of two nucleosides.For purposes of this invention, such a direct 3′-3′linkage (no linkerinvolved) is considered to be a “nucleotidic linkage.”

In some embodiments, the non-nucleotidic linker is a metal, including,without limitation, gold particles. In some other embodiments, thenon-nucleotidic linker is a soluble or insoluble biodegradable polymerbead.

In yet other embodiments, the non-nucleotidic linker is an organicmoiety having functional groups that permit attachment to theoligonucleotide. Such attachment preferably is by any stable covalentlinkage.

In some embodiments, the non-nucleotidic linker is a biomolecule,including, without limitation, polypeptides, antibodies, lipids,antigens, allergens, and oligosaccharides. In some other embodiments,the non-nucleotidic linker is a small molecule. For purposes of theinvention, a small molecule is an organic moiety having a molecularweight of less than 1,000 Da.

In some embodiments, the small molecule is an aliphatic or aromatichydrocarbon, either of which optionally can include, either in thelinear chain connecting the oligonucleotides or appended to it, one ormore functional groups selected from the group consisting of hydroxy,amino, thiol, thioether, ether, amide, thioamide, ester, urea, andthiourea. The small molecule can be cyclic or acyclic. Examples of smallmolecule linkers include, but are not limited to, amino acids,carbohydrates, cyclodextrins, adamantane, cholesterol, haptens andantibiotics. However, for purposes of describing the non-nucleotidiclinker, the term “small molecule” is not intended to include anucleoside.

In some embodiments, the small molecule linker is glycerol or a glycerolhomolog of the formula HO—(CH2) o-CH(OH)—(CH2) p-OH, wherein o and pindependently are integers from 1 to about 6, from 1 to about 4, or from1 to about 3. In some other embodiments, the small molecule linker is aderivative of 1,3-diamino-2-hydroxypropane. Some such derivatives havethe formula HO—(CH2) m-C(O)NH—CH2-CH(OH)—CH2-NHC(O)-m-OH, wherein m isan integer from 0 to about 10, from 0 to about 6, from 2 to about 6, orfrom 2 to about 4.

Modified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. An oligonucleotide is usuallycomprised of more than two (2), and typically more than ten (10) and upto one hundred (100) or more deoxyribonucleotides or ribonucleotides,although preferably between about eight (8) and about forty (40), mostpreferably between about eight (8) and about twenty (20). The exact sizewill depend on many factors, which in turn depends on the ultimatefunction or use of the oligonucleotide. The oligonucleotide may begenerated in any manner, including chemical synthesis, DNA replication,reverse transcription, or a combination thereof.

In the inventive method the oligonucleotides can be administered by anyappropriate administration route, such as, but not limited to,inhalation, ophthalmic, intranasal, parenteral, oral, intradermal andrectal administration. If the patient is also on steroid treatment orother anti-inflammatory treatments such as the use of immunomodulators,the steroids and immunomodulators can be administered together with theoligonucleotides or separately. The route of administration of theoligonucleotides is independent of the route of administration ofsteroids.

The phrase “therapeutically effective amount” as used herein relates toan amount sufficient to enhance steroid efficacy to some beneficialdegree, preferably to enhance by at least about 30 percent, morepreferably by at least 50 percent, and even more preferable by at least90 percent. Most preferably the steroid resistance is treated.

The term “steroid” is used to encompass both corticosteroids andglucocorticosteroids. The term “CG containing oligonucleotide” is usedto encompass an oligonucleotide having at least one unmethylated CGdinucleotide within its entire sequence length and being preferably 8 to100 nucleic acid bases in length.

The expression “enhance steroid efficacy” is here used to encompass asteroid sparing effect, evident as a clinical picture where asimultaneous or sequential treatment with a CG containingoligonucleotide, preferably a pre-treatment, is shown to reduce thesteroid dose necessary to manage inflammation. The expression “enhancesteroid efficacy” is also intended to encompass a synergistic use of aCG containing oligonucleotide and a steroid, either simultaneously orsubstantially simultaneously, or sequentially or substantiallysimultaneously, shown to reduce the steroid dose necessary to manageinflammation. The expressions “steroid resistance” or “steroidrefractory” are used to encompass a patient failing to respondadequately to a current therapeutic regime deemed to be normallyeffective and sufficient to treat the disease in question. Theexpression “steroid dependent” is used to encompass a patient with anobserved inability to be weaned off current therapy without compromisingthe patient status or increasing the severity of the symptoms of thedisease in question.

In one aspect, the invention provides pharmaceutical formulationscomprising an immunomodulatory oligonucleotide, according to theinvention and a physiologically acceptable carrier. As used herein, theterm “physiologically acceptable” refers to a material that does notinterfere with the effectiveness of the immunomodulatory oligonucleotideand is compatible with a biological system such as a cell, cell culture,tissue, or organism. Preferably, the biological system is a livingorganism, such as a vertebrate.

As used herein, the term “carrier” encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, or other materialwell known in the art for use in pharmaceutical formulations. It will beunderstood that the characteristics of the carrier, excipient, ordiluent will depend on the route of administration for a particularapplication. The preparation of pharmaceutically acceptable formulationscontaining these materials are described in, e.g., Remington'sPharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack PublishingCo., Easton, Pa., 1990.

The concentration of an immunomodulating oligonucleotide in apharmaceutically acceptable mixture will vary depending on severalfactors, including the dosage of the compound to be administered, thepharmacokinetic characteristics of the compound(s) employed, the age,sex and condition of the patient, as well as the route ofadministration. Effective amounts of immunomodulating oligonucleotidesfor enhancing steroid efficacy in a steroid resistant or steroiddependent patient would broadly range between about 0.01 μg to about 100mg per kg of body weight, preferably about 0.1 μg to about 10 mg, andmost preferably about 1 μg to about 5 mg per kg of body weight of arecipient mammal.

In certain preferred embodiments, immunomodulatory oligonucleotide,according to the invention are administered in combination with, but notlimited to, anti-inflammatory agents such as TNF-anti-bodies,non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen,naproxen aspirin and other salicylates and cox-2 inhibitors, such ascelecoxib (Celebrex®), corticosteroids (inhaled, oral, rectal), mastcell stabilizers, and leukotriene modifier drugs.

For purposes of this aspect of the invention, the term “in combinationwith” means in the course of treating the same disease in the samepatient, and includes administering the immunomodulatory oligonucleotidein any order, including simultaneous administration, as well astemporally spaced order of up to several months apart. Such combinationtreatment may also include more than a single administration of theimmunomodulatory oligonucleotide. More preferable the immunomodulatoryoligonucleotide of the invention is given to a steroid resistant orsteroid dependent patient after that patient has started steroidtherapy, and is on a stable dosing regime.

In one embodiment the present invention relates to a method forenhancing steroid efficacy in a steroid refractory patient afflictedwith an inflammatory condition not responding or responding poorly orinadequately to anti-inflammatory treatment. An oligonucleotide havingthe sequence formulae:

(SEQ. ID. No. 18) 5′-X_(m)-TTCGT-Y_(n)-3′is administered in an effective amount to the patient, wherein X is A,T, C or G, Y is A, T, C or G, m=0-7, n=0-7 and wherein at least one CGdinucleotide is unmethylated. The oligonucleotide can also have thefollowing formulae:

(SEQ. ID. No. 17) 5′-X_(m)-GTTCGTC-Y_(n)-3′, wherein m = 0-6 and n =0-6; (SEQ. ID. No. 16) 5′-X_(m)-AGTTCGTCC-Y_(n)-3′, wherein m = 0-5 andn = 0-5; (SEQ. ID. No. 15) 5′-X_(m)-CAGTTCGTCCA-Y_(n)-3′, wherein m =0-4 and n = 0-4; (SEQ. ID. No. 14)5′-X_(m)-ACAGTTCGTCCAT-Y_(n)-3′, wherein m = 0-3 and n = 0-3;(SEQ. ID. No. 13) 5′-X_(m)-AACAGTTCGTCCATG-Y_(n)-3′, wherein m = 0-2 andn = 0-2; or (SEQ. ID. No. 12)5′-X_(m)-GAACAGTTCGTCCATGG-Y_(n)-3′, wherein m = 0-1 and n = 0-1;

In one embodiment the oligonucleotide has the formulae:

(SEQ. ID. No. 1) 5′-GGAACAGTTCGTCCATGGC-3′

Oligonucleotides to be used according to the present invention are alsoexemplified in Table 1.

In the method according to the present invention the patient iscurrently on corticosteroid treatment, the patient is steroid dependentand currently on corticosteroid treatment or the patient is currently onanti-inflammatory treatment.

The method according to the invention is for enhancing steroid efficacyin a patient afflicted with an inflammatory condition. The inflammatorycondition is selected from the group consisting of ulcerative colitis(UC), Crohn's disease (CD), rheumatoid arthritis, psoriasis, emphysema,asthma and chronic obstructive pulmonary disease (COPD). In oneembodiment the inflammatory condition is ulcerative colitis and inanother embodiment the inflammatory condition is Crohn's disease.

The oligonucleotide used in the inventive method can be modifiedaccording to methods known for the skilled person and as defined above.For example, at least one nucleotide of the oligonucleotide has aphosphate backbone modification, wherein the phosphate backbonemodification is a phosphorothioate or phosphorodithioate modification.The modification can occur at one or more nucleotides at any positionalong the entire length of the oligonucleotide. In one embodiment thenucleic acid backbone includes the phosphate backbone modification onthe 5′ inter-nucleotide linkages. As an alternative the nucleic acidbackbone includes the phosphate backbone modification on the 3′inter-nucleotide linkages.

In addition to DNA the oligonucleotide can be composed of an analogue ormimic of DNA, including but not restricted to the following:methylphosphonate, N3′->P5′-phosphoramidate, morpholino, peptide nucleicacid (PNA), locked nucleic acid (LNA), arabinosyl nucleic acid (ANA),fluoro-arabinosyl nucleic acid (FANA) methoxy-ethyl nucleic acid (MOE).

Further, the oligonucleotide used in the inventive method can compriseat least one modified sugar moiety nucleobase as defined above. Themodified sugar moiety is, for example, a 2′-O-methoxyethyl sugar moiety.

In one embodiment of the inventive method the oligonucleotide isadministered in combination with corticosteroids.

The present invention also relates to the use of an oligonucleotidehaving the sequence:

(SEQ. ID. No. 18) 5′-X_(m)-TTCGT-Y_(n)-3′for the manufacture of a medicament for enhancing steroid efficacy in asteroid refractory patient afflicted with an inflammatory condition notresponding or responding poorly or inadequately to anti-inflammatorytreatment, wherein X is A, T, C or G, Y is A, T, C or G, m=0-7, n=0-7and wherein at least one CG dinucleotide is unmethlyated.

The oligonucleotides used in the method as defined above can also beused for the manufacture of the medicament.

In the use according to the present invention the patient is currentlyon corticosteroid treatment, the patient is steroid dependent andcurrently on corticosteroid treatment or the patient is currently onanti-inflammatory treatment. In one embodiment the oligonucleotide isadministered in combination with corticosteroids.

The inflammatory condition is selected from the group consisting ofulcerative colitis (UC), Crohn's disease (CD), rheumatoid arthritis,psoriasis, emphysema, asthma and chronic obstructive pulmonary disease(COPD). In one embodiment the inflammatory condition is ulcerativecolitis and in another embodiment the inflammatory condition is Crohn'sdisease.

The immunomodulatory oligonucleotide of the invention is illustrated bySEQ.ID.No 1 (DIMS0150) and serves as an example of immunomodulatory DNAbased oligonucleotides containing a CpG motif. The invention disclosedthe surprising finding that when such immunomodulatory oligonucleotideas denoted by SEQ.ID.NO.1 is administered to a patient suffering from aninflammatory condition of the bowel (i.e ulcerative colitis and Crohnsdisease), and who were equally not responding to steroid therapies andwere on concomitant steroid therapy, there was a rapid and pronouncedimprovement of such patients and the dose of administered steroids couldbe reduced. In contrast, when said immunomodulatory oligonucleotide wasgiven to patients suffering from ulcerative colitis where steroids wereexcluded and the patients were steroid responsive, no improvement intheir disease was seen. This surprising observation clearly indicatedthat through as of yet unknown mechanisms, the immunomodulatory effectsof a CpG containing oligonucleotide in the context of steroid resistanceinduced an improvement of disease that was not apparent in patients thatwere not steroid resistant.

The following non-limiting examples firstly confirm that SEQ.ID.NO.1functions as an immunomodulatory oligonucleotide and that varyinglengths of SEQ.ID.NO.1 retain activity. The latter examples aresummaries of clinical data in patients with ulcerative colitis andCrohns disease receiving a single rectal administration of SEQ.ID.NO.1.

EXAMPLES

Materials and Methods

Oligodeoxynucleotides (ODN).

In the invention numerous ODNs were used for stimulation experimentsusing human peripheral blood monocytes (PBMC) or mouse splenocytes. TheODNs used are listed in Table 1. In some of the oligonucleotides thedinucleotide motif was “reversed”, and as such function as controls

TABLE 1 Immunomodulatory oligonucleotides Compound ID Sequence IDOligo sequence (5′-3′) DIMS0150 SEQ.ID.NO. 1 G*G*A*ACAGTTCGTCCAT*G*G*CIDX0526 SEQ.ID.NO. 2 G*G*A*ACAGTTGCTCCAT*G*G*C IDX0304 SEQ.ID.NO. 3A*G*C*TGAGTAGCCTATA*G*A*C IDX0900 SEQ.ID.NO. 4G*G*TGCATCGATGCAG*G*G*G*G*G IDX0910 SEQ.ID.NO. 5T*C*G*T*C*G*T*T*T*TG*T*C*G*T*T*T*T*G*T*C*G*T*T IDX0915 SEQ.ID.NO. 6T*G*C*T*G*C*T*T*T*T*G*T*G*C*T*T*T*T*G*T*G*C*T*T IDX0250 SEQ.ID.NO. 7G*A*A*ACAGATCGTCCAT*G*G*T IDX0254 SEQ.ID.NO. 8 G*A*A*ACAGATGCTCCAT*G*G*TIDX0920 SEQ.ID.NO. 9 T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T IDX0925SEQ.ID.NO. 10 T*C*C*A*T*G*A*G*C*T*T*C*C*T*G*A*G*C*T*T IDX-0912b1SEQ.ID.NO. 11 T*C*G*TCGTTTTGTCGTTTTGTC*G*T*T IDX-0024b1 SEQ.ID.NO. 12G*A*A*CAGTTCGTCCA*T*G*G IDX-0025b1 SEQ.ID.NO. 12 G*A*ACAGTTCGTCCAT*G*GIDX-0026b1 SEQ.ID.NO. 13 A*A*C*AGTTCGTCC*A*T*G IDX-0027b1 SEQ.ID.NO. 13A*ACAGTTCGTCCAT*G IDX-0028b1 SEQ.ID.NO. 14 A*C*A*GTTCGTC*C*A*TIDX-0029b1 SEQ.ID.NO. 14 ACAGTTCGTCCAT IDX-0030b1 SEQ.ID.NO. 15C*A*G*TTCGT*C*C*A IDX-0031b1 SEQ.ID.NO. 15 CAGTTCGTCCA IDX-0032b1SEQ.ID.NO. 16 A*G*T*TCG*T*C*C IDX-0033b1 SEQ.ID.NO. 16 AGTTCGTCCIDX-0034b1 SEQ.ID.NO. 17 G*T*T*C*G*T*C IDX-0035b1 SEQ.ID.NO. 17 GTTCGTCKey *indicates phosphorothioate linkage while others have phosphodiesterlinkage.Formulation

All ODNs, except for DIMS0150 and IDX0250 synthesized by Avecia, weresynthesized and delivered by Biomers.net, Germany.

The lyophilized ODNs used (see Table 1)—all except human DIMS0150—werefirst diluted in a small volume of distilled water. After thoroughmixing, each ODN was further diluted with water in a series of differentdilutions. The optical density (OD) A260/A280 was determined in at leastfive or more samples of each dilution using a spectrophotometer(SmartSpec 3000, BioRad). The average concentration of all readings, forall dilutions, was calculated in order to determine the concentration ofthe stock. These stock solutions were all stored at −20° C. For allODNs, one portion of the concentrated stock solution was dilutedfurther, in order to obtain one high and one low concentrated stocksolution (1 μg/μl and 20 μg/μl respectively). The concentration wasdetermined in the same manner, measuring OD using a spectrophotometer asmentioned above.

The different working solutions used in the experiments; 0.1 μM, 1 μM, 3μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, 150 μM, 200 μM and 300 μM wereprepared by diluting the ODNs further in PBS using the high concentratedstock solution (20 μg/μl) and the low concentrated stock solution (1μg/μl).

DIMS0150 was diluted in distilled water and the concentration wasdetermined in a similar way as mentioned for the lyophilized ODNs.

Biological Systems

Cell Preparation:

Blood samples were obtained from healthy volunteers. PBMC were isolatedby density gradient centrifugation using Ficoll-Paque Plus (PharmaciaBiotech, Uppsala, Sweden), washed three times in buffered salinesolution (PBS), and resuspended in RPMI 1640 (Sigma) containing 10% heatinactivated fetal calf serum (FCS) (Life Technologies), 100 U/mlpenicillin, 100 μg/ml streptomycin (Life Technologies), 2 mM L-glutamine(Sigma), gentamycin (Sigma) and 5 mM Hepes (Gibco, Life Technologies).Cells were counted using 0, 4% Trypan blue solution (Sigma Aldrich)

Mouse Splenocyte Preparation:

For each experiment a spleen was excised from a C57 BL/6 mouse (micewere ordered from MTC animal unit, Karolinska Institutet) and a singlecell suspension prepared under sterile conditions by using a nylon cellstrainer (Cell strainer 100 μM, BD Falcon). Cells were then washed oncein complete RPMI 1640 (RPMI 1640 containing 5% heat inactivated FCS, 2mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin) at 1200rpm for 7-10 minutes. The supernatant was decanted and cells wereresuspended in 1 ml red blood cell lysing buffer (Sigma) and incubatedfor a maximum of two minutes at room temperature. Another 5 ml completemedium was added before centrifugation performed as previouslydescribed. After decanting the supernatant, cell pellet was resuspendedin complete medium and cell numbers were determined in 0, 4% Trypan bluesolution.

Techniques

ELISpot

PBMC, as described previously, were seeded into a pre-coated, PVDF-basedmembrane plate for ELISpot (MABTech AB, Sweden). Prior to cell additionthe PDVF-plate was coated overnight at +4° C. with a specificcoating-antibody for IFN-alpha, IFN-gamma or IL-10 (included in ELISpotkits; IFN-alpha, IFN-gamma and IL-10 from MABTech AB, Sweden)respectively. PBMC were then seeded at 500 000 cells/well in completeRPMIc. Directly after seeding, cells were treated with the respectiveoligonucleotides (ODN). Each ODN was added to the specific wells givingfinal ODN-concentrations of 0.1, 1, 5, 10, 25, 50, 100, 150 and 200 μMin a total volume of 100 μl/well. Samples were prepared in triplicates.After treatment, cells were incubated in a humified incubator at 5%carbon dioxide at 37° C. IFN-alpha was analyzed for 2, 10 and 3 donorsat 24, 48 and 72 hrs, respectively. IFN-gamma was analyzed for 2, 7 and5 donors at 24, 48 and 72 hrs, respectively. IL-10 was analyzed for 5and 4 donors at 48 and 72 hrs, respectively. Detection and counting ofcytokine producing cells was performed by following the manufacturer'smanual. The ELISpot reader software was AID 2.3.3 located at Center forMolecular Medicine, CMM, Karolinska Hospital, Solna, Sweden.

Enzyme-Linked ImmunoSorbent Assay—ELISA

PBMC, prepared as described previously, were seeded into a 96-well flatbottomed cell culture plate at 500 000 cells/well in RPMIc. Directlyafter seeding, cells were treated with the respective ODN. Each ODN wasadded to the specific wells giving final ODN-concentrations between 0.1,1, 5, 10, 25, 50, 100, 150, 200 and 300 μM in a total volume of 100μl/well. Samples were prepared in duplicates. After treatment, cellswere incubated in a humified incubator at 5% carbon dioxide and 37° C.for 48 hrs. Supernatants were saved and stored at −20° C. prior tocytokine level determination by using specific Quantikine ELISAfollowing the manufacturer's protocol (For human PBMC experiments thefollowing ELISA kits were used: human IL-10 and human IFN-alpha. Formouse splenocytes experiments; murine IL-10; murine IFN-alpha, R&DSystems, Abingdon, UK).

Example 1 Evaluation of Cytokine Production of PBMC Upon Stimulationwith DIMS0150

The immunostimulatory activity of the CpG containing ODN, DIMS0150, wasevaluated in human PBMC. The hypothesis was that PBMC incubated withdifferent concentrations of DIMS0150 for different periods of time wouldstimulate cytokine production in a CpG dependent manner. For thisreason, three cytokines that are well known to be produced by PBMC inresponse to CpG DNA, namely IFN-alpha, IL-10 and IFN-gamma were chosen.Indeed, PBMC from different healthy donors all showed time (data notshown) and dose dependent cytokine production as analysed by ELISpot inresponse to DIMS0150. Among the three cytokines tested, IL-10 was themost responding cytokine after 48 hrs stimulation with DIMS0150 (FIG.1.). In contrast to IL-10, DIMS0150 was less potent at inducingIFN-alpha and IFN-gamma in PBMC at all concentrations and time pointstested, represented by 72 hrs for IFN-gamma (see FIG. 2) and 48 hrs forIFN-alpha (see FIG. 3). A CpG reverted form of DIMS0150, IDX0526, wasalso included in all experiments in order to evaluate the CpG dependencyof potential cytokine production. PBMC treated with the IDX0526 showedno or reduced production of all three cytokines studied compared tostimulation with DIMS0150 (see FIGS. 1, 2 and 3).

Example 2 Quantification of Cytokine Production of PBMC in Response toDIMS0150

In order to quantify the amount of cytokine produced from the positivecells observed by ELISpot, ELISA analyses were performed. PBMC wereincubated with increasing concentrations of DIMS0150 and thesupernatants were analyzed for levels of IL-10, IFN-alpha and IFN-gamma.In agreement with those results obtained by ELISpot data, usingconcentrations between 0.1 and 200 μM (or 300 μM for IFN-alpha andIFN-gamma) resulted in a CpG dependent dose response of all thecytokines after 48 hrs incubation (see FIGS. 4 A, B and C). SinceELISpot and ELISA measure different parameters (i.e. number of cellssecreting a particular cytokine versus the amount of secreted cytokine)the ELISA measurements should be considered as complementary informationregarding the actual amount being produced at an particularconcentration, regardless of the number of cells secreting the cytokineof interest. Thus, the dose response pattern may appear different whencomparing results from those different techniques. The individualvariance in response to DIMS0150 as analysed by quantitative ELISA hasbeen less extensively investigated (1-3 donors), in comparison toELISpot.

Example 3 Comparison of DIMS0150 with Different CpG ODNs in PBMC

A dose response of DIMS0150 stimulation was compared with that of knownhuman and murine CpG ODNs, IDX0910 and IDX0920, respectively. Inaddition, IDX0250 was also included in this experimental set up, sincethis ODN sequence also contains a CpG dinucleotide and may act as CpGDNA. The CpG flanking bases in IDX0250 differ slightly to DIMS0150 andthis may influence the level of cytokine response in PBMC uponstimulation. In this investigation, PBMC were treated for 48 hours withthe CpG ODNs and their respective reversed GpC controls beforesupernatants were analyzed in duplicate using quantitative ELISA assaysfor IL-10 and IFN-gamma DIMS0150 and the IDX0250 gave rise to a similarIL-10 response at 100 μM (FIG. 5.) but at the lowest concentrations (0.1μM to 1 μM), none of these ODNs stimulated IL-10 production of PBMC. Incomparison, PBMC incubation with IDX0910 or IDX0920 reached the highestIL-10 production at the lower concentrations used. IFN-gamma analysis ofthe supernatants resulted in lower secretion of this cytokine comparedto IL-10 (FIG. 6). None of the GpC reversed controls or IDX0304 inducedIFN-gamma but some levels of IL-10 secretion in PBMC were observed withthe two control GpC ODNs, IDX0915 and IDX0925. This may be due to thepresence of a fully phosphorothioate backbone in those ODNs.

Example 4 Comparison of DIMS0150 with Different CpG ODNs in MouseSplenocytes

Humans and mice respond to different CpG ODNs. The immunostimulatoryeffect of DIMS0150 was compared to the same set of CpG ODNs performed inPBMC (see FIG. 6) in a mouse splenocyte system. Splenocytes were treatedwith CpG ODNs and their respective reversed negative GpC control for 48hours before supernatants were analyzed for IFN-gamm

and IL-10 in duplicate using quantitative ELISA assays. Treatment ofsplenocytes with DIMS0150 resulted in a strong IFN-gamma response at thehighest concentration used. However, in this assay IDX0250 was morepotent than DIMS0150, indicating that sequences surrounding the CpG alsohave impact on level of response (FIG. 7). The most pronounced IFN-gammalevels was found in supernatants from cells stimulated with the CpG ODN,IDX0920 at the lower concentrations used. Lastly, analysing thesupernatants for levels of IL-10 (FIG. 8.) showed similar pattern towhat was observed when measuring IFN-gamma. None of the GpC reversed ODNcontrols induced IFN-gamma, but IDX0925 induced some level of IL-10 alsoin the murine system.

Example 5 Inhibition of TLR9

PBMC from healthy volunteers prepared using standard procedure. Cellswere plated onto 96-well cultural plate with density of 5×10⁶ cells perwell in RPMIc containing 5% FCS (Gibco). Chloroquine (CQ) a known TLR9inhibitor and Concavalin A (Con A) were purchased from Sigma, preparedand stored as stock solutions (5 mg/ml). Immunostimulatoryoligonucleotides SEQ.ID.NO.11 (IDX-0912b), SEQ.ID.NO.9 (IDX-0920) andSEQ.ID.NO 1 (DIMS0150) were added to cultivation medium at optimalworking concentrations that were determined previously: 1 uM, 10 uM and100 uM, respectively. Cells were pre-incubated in the cell incubator(37C, 5% CO2) 40 min with 0; 1; 10 or 50 ug/ml CQ. Then IS ODNs wereadded to cultivation medium.

Con A was added to positive control group of cells at finalconcentration of 20 ug/ml.

Three control cell groups were incubated with medium only or mediumcontaining IS ODNs or CQ. 100 ul of supernatant were collected after 24hour of cultivation and used for IL-6 and IL-10 level measurements usingTh1/Th2 CBA kit II (BD). From FIGS. 9A and 9B it can be seen that thereis a dose dependent reduction in levels of IL-10 and IL-6 respectivelywhen stimulated with immunomodulatory oligonucleotides, followingpre-incubation of increasing concentrations of Chloroquine. Theseresults indicate a dependency of TLR9 for the immunomodulatory effectsof the oligonucleotides as determined through cytokine release.

Example 6 Truncation Study

Peripheral blood cell concentrates (buffy coats) were obtained fromhealthy blood donors from the Karolinska University Hospital Blood Bank.Peripheral blood mononuclear cells (PBMCs) were separated onFicoll-Paque (Amersham Biosciences AB, Uppsala, Sweden) by gradientcentrifugation. After washing 3 times in phosphate-buffered saline (PBS,pH 7.4, Ca²⁺ and Mg²⁺ free), the number and viability of the cells weredetermined by Trypan blue exclusion. The cells were diluted to 10×10⁶cells/mL in complete culture medium, RPMIc [RPMI 1640 mediumsupplemented with 25 mg/mL gentamicin, 2 mM L-glutamine, 100 IU/mLpenicillin, 100 mg/mL streptomycin (Gibco BRL, Life Technologies Ltd), 5mM Hepes, and 10% (v/v) heat-inactivated fetal calf serum (FCS, Hyclone,Logan, Utah, USA)]. Isolated PBMCs were cultured in 48-well cultureplates (2×10⁶ cells in 400 μl medium/well) in the presence of differentoligo dinucleotides (ODNs) (Table I) at the final concentration of 10 or100 μM. RPMIc alone served as negative control. The cells were incubatedfor 48 hrs, at 37° C. in a humid condition with 6% CO2 in air. Cellsupernatants were collected and analysed for the presence of cytokine byutilizing the cytometric bead array (CBA) (Becton Dickinson) accordingto the manufacturer's protocol on a FACSCalibur flow cytometer followedby analysis using CellQuest software (Becton Dickinson). The lowerdetection limit was 20 μg/ml for each cytokine. FIGS. 10A and 10B,indicate that decreasing the length of SEQ.ID.NO.1 by truncating thesequence through the removal nucleotides from each end of theoligonucleotide, activity is still retained regarding IL-10 stimulation.For example, IDX-0031b1, a truncated 13 mer of the original SEQ.ID.NO.1,is still able to induce IL-10 at a concentration of 100 uM. At a lowerconcentration of 10 uM, activity is seen up to truncated 15 mer version(IDX-0027b1) from the original SEQ.ID.NO.1.

Example 7 Human Pilot Proof of Concept Study

The Pilot proof of concept study is described in its entirety in AnnexI.

Aims of the Study

Primary objective: To assess the safety issues regarding the use of theDNA based oligonucleotide denoted as SEQ.ID.No 1 in ulcerative colitisand Crohns disease patients.

Secondary objective: To explore the clinical efficacy as determined byendoscopic and clinical remission/improvement rates, histologicalimprovement and changes in clinical laboratory parameters.

The study was placebo controlled; double blinded single dose andconsidered patients that were unresponsive to corticosteroids orcorticosteroid dependent who where on concomitant steroid therapies.

Doses levels used were 3 mg and 30 mg given as a single rectaladministration

Clinical Response at Week 1

i) SEQ. ID. NO. 1 5/7 (71%) responders ii) Placebo ¼ (25%) responders

Overall, this pilot study indicated good efficacy in both dose groupsfollowing a single rectal administration. Perhaps more suspiring was therapidity of response in that all responding patients did so within aweek of receiving the study drug. Of interest was the finding that twofrom the 7 patients that received SEQ.ID.NO.1 are still as of today inremission and steroid free. Moreover, no serious adverse events wererecorded.

Example 8 Clinical Phase II Study

Aims of the Study

Primary objective: To evaluate the ability of each of the four doselevels (0.3 mg, 3 mg, 30 mg and 100 mg) of oligonucleotide SEQ.ID.NO.1as an anti-inflammatory therapy to induce clinical remission in patientswith mild to moderately active ulcerative colitis (UC), as compared withplacebo.

Secondary objective: To assess the tolerability of single rectal dosesof SEQ.ID.NO.1 oligonucleotide and to further evaluate the efficacy andsafety of SEQ.ID.NO.1 oligonucleotide at four dose levels and to assessthe pharmacokinetics of SEQ.ID.NO.1 oligonucleotide after rectaladministration, as compared to placebo.

Study Conclusions

Clinical Response at Week 1, ITT/Safety Population

Clinical 0.3 mg 3 mg 30 mg 100 mg Placebo Response (N = 31) (N = 29) (N= 30) (N = 29) (N = 29) Yes, n (%)  8 (25.8)  6 (20.7)  7 (23.3)  5(17.2) 11 (37.9) No, n (%) 23 (74.2) 23 (79.3) 23 (76.7) 24 (82.8) 18(62.1)

From the table response rate to those receiving active drug was 22% (26/119), placebo was 38% ( 11/29). This study could not confirm that onesingle dose of SEQ.ID.NO.1 oligonucleotide in doses from 0.3 to 100 mgin a limited number of patients, can induce clinical, endoscopic orhistopathological remissions or responses over a 12 week period,however, this study demonstrated a good safety profile of the drug.

In Comparison Clinical Response Rates at Week 1

Pilot study Phase II Active 71% 22% Placebo 25% 38%

It is apparent that patients from the pilot study had a much betterresponse rate than that seen in phase II. It is also clear that whilepatients from the pilot study where allowed steroids as concomitantmedications and where resistant or dependent on corticostroids, it wasan exclusion criteria in phase II. No steroids were allowed during theduration of the phase II study and the patients were neither resistantnor dependent on steroid therapies.

The diverging results between the pilot study and larger phase II studywould suggest that patients that are resistant to or dependent oncorticosteroids and on concomitant corticosteroid therapy respond morefavourably to a single rectal dose of SEQ.ID.No 1 than those patientsthat are not. The reason for this surprising difference in clinicaloutcome is not clear. However, the immunomodulating action of CpGcontaining oligonucleotides could induce beneficial changes to thepatient's immune system such that steroid resistant or steroid dependentpatients were able to respond to steroids again. In other words,immunomodulating oligonucleotides may induce a re-sensitization of thepatients to the anti-inflammatory effects of steroids.

The provided examples confirm that immunomodulatory oligonucleotidesthat contain a CpG dinucleotide within their sequence such as exampleSEQ.ID.NO.1 are able to induce certain cytokines for which there existsevidence of their role in modulating steroid responsiveness, asmentioned in background art. In light of such, immunomodulatoryoligonucleotides that induce the production of interferons and IL-10,for example, may prove beneficial.

Although particular embodiments have been disclosed herein in detail,this has been done by way of example for purposes of illustration only,and is not intended to be limiting with respect to the scope of theappended claims that follow. In particular, it is contemplated by theinventor that various substitutions, alterations, and modifications maybe made to the invention without departing from the spirit and scope ofthe invention as defined by the claims.

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What is claimed is:
 1. A method for enhancing steroid efficacy in apatient afflicted with an inflammatory condition and currently on andresponding adequately to steroid anti-inflammatory treatment, with theinability to be weaned off systemically or topically administeredsteroid treatment without increasing severity of the inflammatorycondition, comprising: administering to the patient an oligonucleotidehaving the sequence 5′-TTCGT-3′ (SEQ ID NO: 18) and not more than 100nucleotides, and wherein at least one CG dinucleotide is unmethylated,wherein the oligonucleotide is administered in an amount effective toimprove sensitivity of the patient to the steroid anti-inflammatorytreatment and thereby enable the patient to be weaned off of the steroidanti-inflammatory treatment.
 2. The method according to claim 1, whereinthe oligonucleotide has not more than 40 nucleotides.
 3. The methodaccording to claim 1, wherein the oligonucleotide has not less than 8nucleotides.
 4. The method according to claim 1, wherein theoligonucleotide has between 8 and 40 nucleotides.
 5. The methodaccording to claim 1, wherein the oligonucleotide has the sequence5′-Xm-TTCGT-Yn-3′ (SEQ ID No. 18) wherein X is A, T, C or G, Y is A, T,C or G, and m+n is not greater than 95, and wherein at least one CGdinucleotide is unmethylated.
 6. The method according to claim 1,wherein the oligonucleotide has the sequence 5′-Xm-TTCGT-Yn-3′ (SEQ IDNo. 18) wherein X is A, T, C or G, Y is A, T, C or G, and m+n is notgreater than 35, and wherein at least one CG dinucleotide isunmethylated.
 7. The method according to claim 1, wherein theoligonucleotide has the sequence 5′-Xm-TTCGT-Yn-3′ (SEQ ID No. 18)wherein X is A, T, C or G, Y is A, T, C or G, m=0-7, and n=0-7, andwherein at least one CG dinucleotide is unmethylated.
 8. The methodaccording to claim 7, wherein the oligonucleotide is 5′-Xm-GTTCGTC-Yn-3′(SEQ ID No. 17) and wherein m=0-6 and n=0-6.
 9. The method according toclaim 7, wherein the oligonucleotide is 5′-Xm-AGTTCGTCC-Yn-3′ (SEQ IDNo. 16) and wherein m=0-5 and n=0-5.
 10. The method according to claim7, wherein the oligonucleotide is 5′-Xm-CAGTTCGTCCA-Yn-3′ (SEQ ID No.15) and wherein m=0-4 and n=0-4.
 11. The method according to claim 7,wherein the oligonucleotide is 5′-Xm-ACAGTTCGTCCAT-Yn-3′ (SEQ ID No. 14)and wherein m=0-3 and n=0-3.
 12. The method according to claim 7,wherein the oligonucleotide is 5′-Xm-AACAGTTCGTCCATG-Yn-3′ (SEQ ID No.13) and wherein m=0-2 and n=0-2.
 13. The method according to claim 7,wherein the oligonucleotide is 5′-Xm-GAACAGTTCGTCCATGG-Yn-3′ (SEQ ID No.12) and wherein m=0-1 and n=0-1.
 14. The method according to claim 7,wherein the oligonucleotide is 5′-GGAACAGTTCGTCCATGGC-3′ (SEQ ID No. 1).15. The method according to claim 1, wherein said patient is currentlyon corticosteroid anti-inflammatory treatment.
 16. The method accordingto claim 1, wherein the inflammatory condition is selected from thegroup consisting of ulcerative colitis (UC), Crohn's disease (CD),rheumatoid arthritis, psoriasis, emphysema, asthma and chronicobstructive pulmonary disease (COPD).
 17. The method according to claim1, wherein the inflammatory condition is ulcerative colitis.
 18. Themethod according to claim 1, wherein the inflammatory condition isCrohn's disease.
 19. The method according to claim 1, wherein saidoligonucleotide comprises at least one nucleotide having a backbonemodification.
 20. The method according to claim 19, wherein saidoligonucleotide comprises at least one nucleotide having a phosphatebackbone modification.
 21. The method according to claim 20, wherein thephosphate backbone modification is a phosphorothioate orphosphorodithioate modification.
 22. The method according to claim 20,wherein the phosphate backbone modification is on the 5′inter-nucleotide linkages.
 23. The method according to claim 20, whereinthe phosphate backbone modification is on the 3′ inter-nucleotidelinkages.
 24. The method according to claim 20, wherein the phosphatebackbone modification is on the 5′ inter-nucleotide linkages and the 3′inter-nucleotide linkages.
 25. The method according to claim 19, whereinthe modification occurs at one or more nucleotides at any position alongthe entire length of said oligonucleotide.
 26. The method according toclaim 1, wherein said oligonucleotide is an oligonucleotide composed ofDNA or an analogue or mimic of DNA.
 27. The method according to claim26, wherein said oligonucleotide is an oligonucleotide composed of DNAor an analogue or mimic of DNA selected from the group consisting ofmethylphosphonate, N3′->P5′-phosphoramidate, morpholino, peptide nucleicacid (PNA), locked nucleic acid (LNA), arabinosyl nucleic acid (ANA),fluoro-arabinosyl nucleic acid (FANA) methoxy-ethyl nucleic acid (MOE).28. The method according to claim 1, wherein said oligonucleotidecomprises at least one modified sugar moiety nucleobase.
 29. The methodaccording to claim 28, wherein the modified sugar moiety is a2′-O-methoxyethyl sugar moiety.
 30. The method according to claim 1,wherein the amount of oligonucleotide administered to the patient isabout 0.01 μg to about 100 mg per kg body weight.
 31. The methodaccording to claim 30, wherein the amount of oligonucleotideadministered to the patient is about 0.1 μg to about 10 mg per kg bodyweight.
 32. The method according to claim 30, wherein the amount ofoligonucleotide administered to the patient is about 1 μg to about 5 mgper kg body weight.
 33. The method according to claim 1, wherein theoligonucleotide is administered via inhalation, or opthalmically,intranasally, parenterally, orally, intradermally, or rectally.
 34. Themethod according to claim 1, wherein the patient is human.
 35. Themethod according to claim 1, wherein the oligonucleotide is administeredas a single administration.
 36. The method according to claim 1, whereinthe oligonucleotide is administered in more than a singleadministration.